Tuberculin active proteins and peptides from the cells of tubercle bacilli

ABSTRACT

Tuberculin active proteins and peptides which have the following linkage

United States Patent Tsumita et al.

[ June 10, 1975 TUBERCULIN ACTIVE PROTEINS AND PEPTIDES FROM THE CELLS OF TUBERCLE BACILLI Inventors: Toru Tsumita; Seishi Kuwabara, both of Tokyo, Japan Assignee: Mitsui Pharmaceuticals,

Incorporated, Tokyo, Japan Filed: June 28, 1973 Appl. No.: 374,663

References Cited OTHER PUBLICATIONS Rzucidlo et al.: Chem. Abstr., 72, 29006d (1970).

Daniel et al.: Chem. Abstr., 72, 107l28m (1970).

Stoeckl et al.: Chem. Abstr. 60, 9763g (1964).

Rhodes: Chem. Abstr. 56, 9204e (1962).

Primary ExaminerLewis Gotts Assistant Examiner-Reginald J. Suyat Attorney, Agent, or Firm-Fisher, Christen & Sabol Tuberculin active proteins and peptides which have the following linkage ABSTRACT as the active functional group and a process for preparing thereof from the cells of tubercle bacilli.

4 Claims, N0 Drawings TUBERCULIN ACTIVE PROTEINS AND PEPTIDES FROM THE CELLS OF TUBERCLE BACILLI The present invention is concerned with pure tuberculin proteins and peptides which have the linkage AspGlySerGluMet as the active functional group.

It is well known that purified protein derivative (PPDs) purified from extracellular old tuberculin (OT) have been used exclusively for the diagnosis of tuberculosis. Unfortunately, in addition to tuberculin protein, PPDs contains other inactive proteins, nucleic acids, polysaccharides and fatty acids. As pure tuberculin proteins have not been obtained, their physicochemical properties and amino acid sequences have not been determined.

The present inventors have succeededin isolating tuberculin proteins from the cells of tubercle bacilli and have obtained them as crystalline forms. Subsequently the amino acid sequences of the crystalline tuberculin proteins have been determined. Besides, they have obtained the active peptides by hydrolyzing the crystalline tuberculin proteins.

From the results of the investigation, it has been found that the tuberculin activity is closely connected with the aforesaid functional group in proteins and peptides.

The tuberculin proteins of the present invention are prepared from the cells of tubercle bacilli by treating with an organic solvent to obtain water-soluble proteins. The active proteins are mainly purified onto DEAE-cellulose and Sephadex by column chromatography.

The materials used in the present invention are obtained from human-, bovineand avian-type Mycobacterium tuberculosis including BCG and others. The crystalline tuberculin protein from human type Mycobacterium tuberculosis strain Aoyama B is composed of 89 amino acid residues per molecular or 9,700g. On the other hand, the active protein from BCG is composed of 135 amino acid residues per molecular or 13,500g. The obtainable tuberculin proteins depend upon the starting materials used in the present method.

The organic solvents used for purification are acetone, methanol or acetic acid. Another organic solvent, such as ketone, alcohol or carboxylic acid is also employed. These solvents may, of course, be used with water.

The materials without lipo-soluble component are dialyzed against buffers, pH 5.5-7.8 such as tris(hydroxymethyl)aminornethanehydrochloric acid buffer, pH 7.0 containing ethylenediamine tetraacetic acid, or sodium phosphate, dibasic/pottasium phosphate, monobasic buffer, pH 7.2 containing ethylenediamine tetraacetic acid, and the like.

The dialyzed proteins are purified onto column chromatography, two dimensional high' voltage paper electrophoresis followed by paper chromatography. Especially, column chromatography or gel filtration is preferably used, such as diethylaminoethyl(DEAE)- cellulose, aminoethyl(AE)-cellulose, ECTEOLA- cellulose, Sephadex (3-150 or Sephdex 6-200 column. The dialyzed proteins are eluted with the aforesaid buffers.

The crystalline tuberculin proteins of the present invention are soluble in water and their molecular weights 3,000-35,000 are depending upon the starting materials. Tuberculin skin tests indicate that the tuberculin proteins of the present invention are l00 times potent that that of PPDs against guinea pigs sensitized with heat-killed Mycobacterium tuberculosis strain Aoyama B or BCG.

The tuberculin peptides of the present invention are prepared by hydrolyzing the above crystalline tuberculin proteins and are obtained by fractionating their hydrolysates by chromatography.

The tuberculin proteins may be hydrolyzed chemically or enzymatically. The chemical hydrolysis is carried out with a mineral acid. The enzymatic hydrolysis is carried out with a proteolytic enzyme such as trypsin, chymotrypsin, pepsin, papain, bacterial proteinases, carboxypeptidase A or B, and the like.

The resulting hydrolysates are fractionated with column chromatography, paper chromatography, high voltage paper electrophoresis, gel filtration, and the like.

The gel filtration is carried out by elution with an ammonium acetate buffer, pH 5.9 through Sephadex G25 or CM-Sephadex, with an ammonium acetate buffer, pH 4.5 through SE-Sephadex C-50 or with a pyridine-acetic buffer, pH 3.1 through Dowex 50-X2 or lX2.

The tuberculin peptides of the present invention are white crystalline solids, soluble in water and about 500-3000 in the molecular weights.

Tuberculin skin tests indicate that the tuberculin peptides of the present invention show appreciable values of the of PPDs against guinea pigs sensitized with heat-killed Mycobacterium tuberculosis strain Aoyama B or BCG.

The following examples illustrate preparation and purification of the tuberculin proteins and peptides of the present invention.

EXAMPLE 1 The cultured cells (g) of Mycobaceterium tuberculosis strain Aoyama B were killed by heating at C for 30 minutes. The cells were separated from the culture medium by filtration, then sonically disrupted and treated with acetone. The resultant residue was dialyzed against 0.001M tris(hydroxymethyl)aminomethanehydrochloric acid buffer, pH 7.0 containing 0.0001 M ethylenediamine tetraacetic acid.

Dialyzed residues were chromatographed on DEAE- cellulose. Elution was carried out in a sodium chloride gradient. Virtually all of the tuberculin activity was found in the second protein fraction. Further chromatography on Sephadex G200 was carried out in the same buffer as before and fractionated into three fractions.

Nearly all of the active material was found in the major protein fraction. The active material crystallized spontaneously when the solution was kept at 4C in the mixed solution of the same buffer with purified acetone.

From 100g cells (wet weight) of Mycobacterium tuberculosis strain Aoyama B, 20mg of crystalline tuberculin protein was obtained.

The tuberculin protein thus obtained was composed of 89 amino acid residues and its amino acid sequence was determined as follows.

12 3 4 5 6 7 8910 H N-Arg-Leu-Leu-Asp-Asp-Thr-Pro-Glu-Val-Lys- Cys. .Cys. .AspG1ySer-G1uMet-. .Ala-Lys-COOH Tuberculin skin test showed that this tuberculin protein was 100 times 'potent than that of PPDs against guinea pigs sensitized with heat-killed strain Aoyama B or BCG.

EXAMPLE 2 guinea pigs sensitized with" heat-killed Aoyama B'or solution. The resultant hydrolysates were chromatow graphed on column of Sephadex (Ti-25, elution being carried out with ammonium acetate buffer, pH 5.9. The tuberculin active peptides thus obtained was further fractionated with pyridineacetic acid buffer, pH 3.1 on Dowex .lX2 column.

The tuberculin pentapeptide thus obtained was the same as in Example 3.

EXAMPLE 5 The tuberculin protein in Example 2 was treated the same as before. The same tuberculin pentapeptide as in Example 3 was also obtained.

What is claimed is:

' 1. A tuberculin active simple protein from Mycobacterium tuberculosis strain Aoyama B which is composed of 89 amino acid residues and whose amino acid se- 25 quence is as follows BCG.

1 2 3 4 5 6 7 8 9 10 H N-Arg-Leu-LeuAsp-Asp-Thr-Pro-Glu-Val-Lysl' '1 27 68 70 71 72 73 74 88 89 Cys .Cys .Asp-Gly-Ser-Glu-Met- .Ah-Lys-COOH- EXAMPLE 3 several active peptides. One of them was a pentapeptide having' the following'a'mino acid sequence.

'5 '2 3 4 5 H NAsp-Gly-Ser-Glu-Met-COOH 5 Another wasan oligopeptide having the molecular weight of about 950.

Tuberculin skin tests showed that these tuberculin peptides had 1-10 percent activities of the starting tuberculin protein.

EXAMPLE 4 The crystalline tuberculin protein obtained in Example 1 was hydrolyzed in the 0.5 percent chymotrypsin 6 2; A tuberculin active pentapeptide whose amino 5 acid sequence is as follows 1 2 3 4 5 H NAsp-G1y-Ser-Glu-M et-COOH 3, A process for preparing tuberculin active simple proteins having the following tuberculin active group which comprises (a) treating Mycobacterium tuberculosis strain Aoyama B with an organic solvent, (b) dialyz ing the resultant material against a buffer of pH 5.5-7.8 containing ethylenediamine tetraacetic acid and (c) fractionating the resultant dialysate by chromatography and/or gel filtration.

4. A process for preparing tuberculin active simple proteins having the following tuberculin active group which comprises (a) treating BCG with an organic solvent, (b) dialyzing the resultant material against a buffer of pH 5.5-7.8 containing ethylenediamine tetraacetic acid and (c) fractionating the resultant dialysate by chromatography and/or gel filtration. 

1. A TUBERCULIN ACTIVE SIMPLE PROTEIN FROM MYCOBACTERIUM TUBERCULOSIS STRAIN AOYAMA B WHICH IS COMPOSED OF 89 AMINO ACID RESIDUES AND WHOSE AMINO ACID SEQUENCE IS AS FOLLOWS H2N-ARG-LEU-LEU-ASP-ASP-THR-PRO-GLU-VAL-LYS...CYS... CYS...ASP-GLY-SER-GLU-MET...ALA-LYS-COOH.
 2. A tuberculin active pentapeptide whose amino acid sequence is as follows
 3. A process for preparing tuberculin active simple proteins having the following tuberculin active group -Asp-Gly-Ser-Glu-Met-which comprises (a) treating Mycobacterium tuberculosis strain Aoyama B with an organic solvent, (b) dialyzing the resultant material against a buffer of pH 5.5-7.8 containing ethylenediamine tetraacetic acid and (c) fractionating the resultant dialysate by chromatography and/or gel filtration.
 4. A process for preparing tuberculin active simple proteins having the following tuberculin active group -Asp-Gly-Ser-Glu-Met-which comprises (a) treating BCG with an organic solvent, (b) dialyzing the resultant material against a buffer of pH 5.5-7.8 containing ethylenediamine tetraacetic acid and (c) fractionating the resultant dialysate by chromatography and/or gel filtration. 